Assay of blood or other biologic samples for target analytes

ABSTRACT

A patient&#39;s health may be diagnosed by centrifuging blood samples in a transparent tube, which tube contains one or more bodies or groups of bodies such as floats, inserts, liposomes, or plastic beads of different densities. Each density-defined body carries analyte-capture binding materials such as antigens or antibodies, which are specific to an epitope, or other specific high affinity binding site on a target analyte which target analyte may be in the blood or other sample being tested; and the level of which analyte is indicative of the patient&#39;s health. At least one labeled binding material which is also specific to an epitope, or other specific high affinity binding site on the target analyte is added to the sample so as to form labeled binding material/analyte/body complexes in the sample. Upon centrifugation, the complexes will settle out in different areas in the tube according to the respective density of the body or bodies; and the degree of label emission of the complex layers can enable qualitative and/or quantitative analyses of the sample to be made. Unbound labeled binding materials will be separated from the complexed layers by the washing action of ascending or descending components of the sample during the centrifugation step. Unbound labeled binding material will thus not interfere with the analysis.

This is a division of U.S. Ser. No. 08/247,336, field May 23, 1994, nowU.S. Pat. No. 5,635,362 which in turn is a continuation-in-part of U.S.Ser. No. 07/969,379 filed Oct. 30, 1992, now U.S. Pat. No. 5342,790".

TECHNICAL FIELD

This invention relates to the one-step simultaneous determination of thepresence or absence of a target analyte or analytes, and where desired,analyte quantification in a whole blood, anticoagulated whole blood,blood plasma, blood serum, saliva, cerebrospinal fluid, lymph, urine, orother biological fluid sample. More particularly, this inventioninvolves the use of target analyte-capture bodies which are caused tosettle into a predetermined location in a transparent tube, and alsoinvolves labeling of the captured analyte.

BACKGROUND ART

Analyses of blood samples for the presence or absence of antibodies orantigens are used in the diagnosis of diseases, such as HIV infection,hepatitis, Lyme disease, prenatal profiles including TORCH (an acronymfor: "Toxoplasmosis, Rubella, Cytomegalovirus, Herpes") profiles, aswell as other infectious disease profiles. Presently, such serologicdiagnoses are often performed by fluorescent immunoassay.Immunofluorescent assays are not only used for detection of antigens andantibodies in infectious diseases but are widely used for detection ofautoimmune phenomena and for localizing pathologically altered serum ortissue elements, e.g., Graves disease, connective tissue diseases,multiple sclerosis, kidney disease, myasthenia gravis, pemphigus,pemphigoid, as well as many pathologic conditions such as cirrhosis ofthe liver, vasculitis etc. Analyses may also be performed to measure thelevels of hormones such as insulin, thyroxine, thyroid stimulatinghormone (TSH), blood coagulation factors, factor VIII, von Willebrandfactor; and levels of other possible blood constituents, such asdigoxin, morphine, and also blood vitamins such as B₁₂ and folic acid;as well as any other substances which may be present in small amounts inthe biologic sample, which substances have a specific binder available.

Non-immunologic binding pairs include systems wherein the two componentsshare a natural affinity for each other but are not antibodies.Exemplary non-immunologic pairs are biotin/streptavidin orbiotin/avidin; intrinsic factor/vitamin B₁₂ ; folic acid/folate bindingprotein; hormone/hormone receptor; nucleic acid complexes;antibody/antibody binding protein, i.e., IgG/protein A; DNA/DNA;DNA/RNA/; carbohydrates/lectins; complementary peptide sequences;complementary nucleotide sequences; effector/receptor molecules; enzymecofactors/enzymes; enzyme inhibitors/enzymes; a peptidesequence/antibodies specific for that sequence protein; polymericacids/bases; dyes/protein binders; peptides/specific protein binders;and the like.

In a standard indirect fluorescent immunoassay, an antigen, which is thecoupling partner for the antibody to be detected, i.e. float tissueculture cells for DNA antigen, or tissue (skin, liver, muscle, etc) orany microorganism or virus, is first affixed to a solid support mediumsuch as a glass slide. With soluble antigens, a paper membrane,nitrocellulose or nylon membrane (Dot blot Technique) can be used. Asample of serum from the patient is then allowed to incubate in contactwith the affixed antigen for a period of time sufficient for the partnerantibody, if present, to become attached to the affixed antigen. Thesupport surface is then washed to remove all unbound antibodies. Areagent consisting of a labelled antibody to human immune (antibody)globulins is next brought into contact with the support surface andincubated for a time sufficient to cause linkage of the labelledmaterial and any traces of the patient's antibodies which may have boundto the fixed antigen. The excess reagent is then washed off and thesupport surface is examined to determine if any label is present.Examination of the prepared sample, depending upon the nature of thelabel, can be done visually; or by spectrophotometry or fluorometry; orby radiation detection means.

It will be appreciated that the aforesaid procedure requires multiplespecimen handling steps, including washing, and analysis techniques, andis thus labor intensive and time-consuming. The aforesaid procedure isusually used to detect the presence or absence of only oneantigen-specific antibody per test but under certain circumstances twodifferent fluorescent dye labels (fluorescein and rhodamine) are used ifone suspects that at least two antibodies may be implicated andcharacteristic in the suspected disease, i.e., basement membraneantibody as well as DNA antibody, and the substrate used is, forexample, a skin section. Usually an antiglobulin is used as the secondantibody, however if it is desired to ascertain if the antibody presentin the body fluid is IgG or IgM, this can be done using specificanti-IgG or IgM or for that matter any anti-immunoglobulin. Specificimmunoglobulins are often used to determine placental transfer ofdisease, such as syphilis, viral infections, etc, in order to ascertainif the disease is in early or late phases, or to determine whether apatient is producing a specific immunoglobulin to an antigenic stimulus.Fluorescent immunoassays for hormones, drugs and proteins of diagnosticimportance have also been developed.

DISCLOSURE OF THE INVENTION

Red cells, when centrifuged in a tube containing a whole blood samplewill form a continual density gradient layer in the bottom of the tube,with the most dense red cells settling on the bottom of the red celllayer. When an anticoagulated whole blood sample is centrifuged in atube containing bodies of different specific gravity, such ascylindrical floats, beads, discs, or different specific gravityliposomes, these bodies will form spaced, distinctly visible markers inthe packed red cell layer. In certain cases, the centrifuge tube mayalso contain a cylindrical plastic float or insert which may be fixed tothe bottom of the tube or may be freely movable in the tube, and which,if freely movable, has a specific gravity such that it will preferablysink through the red cell layer in the centrifuged blood sample, or cometo rest in an area where the particles also come to rest. The insertrestricts the available space in the tube which the red cells, plasma,serum, or liquid phase can occupy, and therefore increases the distancebetween the marker rings which form in the centrifuged red cell, plasma,serum, or liquid phase layer, and displaces the beads or liposomes tothe periphery of the tube where they may be seen and easily detectedwithout their signal being extinguished by the red cells or serum.Specimens such as plasma or serum can also be analyzed for the presenceof target analytes by using analyte-capturing float/inserts ormicroparticles of one or more characteristic densities coupled withcapture binding material, so that bands of the captured analytes coupledwith labeled binding material will settle into a restricted space on orbetween the float/insert or post in the tube, where the captured analytebands can be observed and measured.

In one embodiment of the invention, the insert itself can be coated withthe capture material, and can be centrifuged back and forth through thetube several times so that the cellular and other components of thesample are forced to pass between the insert and the tube bore wallseveral times. Due to the severely restricted space available forpassage of the sample components between the insert and the tube, whichspace may be in the range of twenty to several hundred microns, and willtypically be in the range of thirty five to forty five microns, theefficiency and dynamics of target analyte capture by the insert aresignificantly enhanced in certain cases.

In performing the method of this invention, density markers such as, forexample, beads, discs, a float/insert (or float/inserts), or liposomes,will be coupled with a capture binding material such as antigens,antibodies, nucleotides, or other active binding substances whosecomplement, or binding partners, (which are designated herein as "targetanalytes") may be present in the patient's blood or other biologicsample. Examples of biologically active complementary couples include:enzymes and their substrates; nucleotides and their complementarynucleotides; aptamers (nucleotides which bind to a target analytemolecule) and their complementary molecules; naturally occurring proteinbinders, such as thyroid binding globulin (TBG) and thyroxine; the"intrinsic factor", and vitamin B-12; CD-4 antibody, or other specificbinding substance affinity binding site and CD-4positive cells and/orany antibody, or other specific binding substance or cells havingepitope, or any specific high affinity binding sites, bound by thatantibody, or other specific binding substance; and specific antibodieswhich will selectively couple with RNA-DNA hybrids, as described byStollar and Rashtchian, in their article "Immunochemical Approaches toGene Probe Assays", Analytical Biochemistry 1987; 161, 38714 394.

Each density-marker body, or group of bodies, of which there may be oneor more, will be coupled with a capture binding material, which isspecific to a target analyte, which analyte may be present in the bloodor other biological specimen sample. The sample is added to the tube soas to allow the density-marker/binding material capture body or bodiesto incubate and intermix with the sample sufficiently to cause anytarget analytes present in the sample to couple with and be captured bytheir complementary partners on the density-markers. In certain cases,the sample-capture body-label incubation may take place during thecentrifugation procedure, particularly when the latter involves reversecentrifugation of the insert back and forth through the tube. Thus theincubation step may actually take place in the centrifuge tube. One ormore labeled antibodies, or other binding material moieties which may bespecific to the same or to a different epitope, or any specific highaffinity binding site on the target analyte, may be used to bind to, andthus label, all density-markers which have a target analyte bondedthereto.

The label may be a liposome encapsulated colorant, or a fluorescentcolorant; or may be a radioactive energy emitter. The label must bedetectable and preferably quantifiable. After an appropriate incubationperiod, the sample is centrifuged to densimetrically separate thedensity-markers into spaced-apart bands or rings in the tube. Thedifferent density-marker bands are then examined in the tube todetermine which bands, if any, have a detectable quantity of label, andto measure the quantity of the label, if appropriate. Quantification anddetection of false positive tests could be furthered by carrying outsuitable control tests. The label most likely to be used would be afluorescent molecule such as FITC (fluorescein isothiocyanate).

The types of label markers (or labels) which are employed withimmunodiagnostic assays can be varied and can include labels which areobserved directly, such as fluorescein; or observed after a furtherstep, such as enzymes. Any suitable label may be employed. Examplesinclude: enzyme labels such as alkaline phosphatases; peroxidases; acidphosphatases; chemiluminescent materials; bioluminescent materials;fluorescent labels; and electron dense labels. Typically, the label is aprotein. The labels may be attached to the target analyte by anysuitable technique.

If desired, the different density beads can have different intrinsiccolors, so that each (if there are more than one band) differentlycolored band will designate a different target analyte. If differentlycolored labeled binding material moieties are used, the colors of thelabeled bands in the tube will indicate which bound analytes are in thesample, and which analytes are not, in the event that bands ofdensity-markers placed in the tube do not demonstrate any labelassociated therewith. This information, of course, permits diagnosis ofthe health of the sample donor in regard to the parameters beingevaluated. The assay will provide the physician with a valuable adjunctto conventional diagnostic tests and will permit the rapid acquisitionof diagnostic data and the rapid screening and identification ofelements sought after in the sample.

In accordance with one embodiment of the invention, a fluorescent assayof a whole blood or blood product sample can be made in a tube andfloat/insert paraphernalia sold by Becton Dickinson and Company underthe trademark QBC®. The labelled binding particle is a particle which isspecific against the same or a second epitope, or any specific highaffinity binding site on the target analyte. The density markerparticles may be differentially colored, one density from another, andthe labelled binding particles may also be differentially colored, onefrom another.

In cases where it is desired to perform one or more simultaneous assaysin the same QBC® tube, the labeled binding materials'tag or label may beidentical, since the labeled binding material will be detected by eitherits position in the tube, which will be a function of the density of theparticle or float/insert to which it is attached, or the position of thelabeled binding material on the float/insert, if the capture bindingmaterial is coated circumferentially around a float/insert in a band orbands, with multiple capture binding material bands being positioned onthe float, which bands are separated by discernible distances on thefloat/insert. As previously noted, the labeled binding materials mayalternatively be provided with different detectable labels. By measuringthe amount of each label, one can quantify the amount of target analytepresent in the sample, since the intensity of the label signal will beproportional to the amount of labeled analyte in the specimen sample.

A variety of immunodiagnostic assays are known to persons of ordinaryskill in the art and can be adapted for use with this invention. Forexample, a target analyte can be detected in accordance with the presentinvention in a competitive assay. In a competitive assay, a labeledmarker competes with unknown sample analyte for a limited number ofcomplementary specific binding pair member sites immobilized on a solidphase support, such as a sphere, liposome, float/insert, or the like.The detectable marker that is bound and quanntified is inverselyproportional to the unknown analyte.

It is therefore an object of this invention to provide an improvedtechnique for analyzing a biological specimen sample to determine thepresence or absence of certain target analytes therein.

It is a further object to provide an improved technique of the characterdescribed wherein the analysis is performed densimetrically in atransparent specimen tube.

It is an additional object of this invention to provide an improvedtechnique of the character described wherein the analysis is performedby using spatially resolvable markers which may have different specificgravities, and which are coupled to capture materials such as antibodiesand/or antigens so that one or more of different multiple assays may beperformed in one tube at the same time.

It is another object of this invention to provide an improved techniqueof the character described wherein the analysis is performed by forming,for example, highlighted antibody/antigen couple bands in the sample.

Still a further object of the invention is the elimination of many ofthe tedious procedural steps such as washing, agitation, preparation ofnumerous reagents such as buffers, blockers and absorbents etc. whichare needed to perform numerous titrations of reagents necessary in priorart assay procedures.

Another object is the elimination of time-consuming procedures such aslengthy incubation steps, as for example are necessary in the ELISAassay often used in similar applications.

Another object of this procedure is the decreased use of radioisotopesand the special apparatus necessary for handling such samples andreagents.

This invention allows for the determination and/or quantitation of verysmall amounts of analyte, using very small amounts of biologic sampleand reagents. A number of important advantages of this procedure arethat this assay can help establish if a disease pathology is the resultof an allergic mechanism, e.g. rheumatoid or connective tissue diseases;establish the specific organism responsible for a disease; establish thecourse of a disease by following antibody, or other specific bindingsubstance titres, which this test can readily do; determine whether aninfant has been infected in utero; and the like.

These and other objects and advantages will become more readily apparentfrom the following detailed description of a preferred embodiment of theinvention when taken in conjunction with the accompanying drawings inwhich:

DESCRIPTION OF THE DRAWING

FIG. 1 is side elevational view of a centrifuge tube adapted to performthe procedure of this invention;

FIG. 2 is a view of the tube of FIG. 1 showing a centrifuged whole bloodsample therein, and with the red blood cell layer being blown up orincreased in size to particularly point out the nature of the invention;

FIG. 3 is an axial sectional view of a second embodiment of a centrifugetube adapted for use in performing the invention;

FIG. 4 is a side elevational view of a float/insert with a number ofspaced-apart antibody, or other specific binding substance bands coatedthereon, along the axial length thereof

FIG. 4A is a view similar to FIG. 4, but showing an embodiment using twofloat/inserts, one for analyte analysis, and the other for a secondanalyte or for other purposes;

FIG. 5 is a schematic representation of the invention in use to detectthe analyte TSH (thyroid stimulating hormone) in a blood sample;

FIG. 6 is a fragmented side elevational view of an embodiment of theinvention which can be used with a centrifuged sample of the typedescribed above, or with a sample which displaces the target analyte ina blood sample below the red cells in a centrifuged blood sample; and

FIG. 7 is a schematic representation similar to FIG. 5, but showing howthe invention can be used to detect analyte cells in a fluid sample.

DETAILED DESCRIPTION OF THE INVENTION

Referring now to FIGS. 1 and 2, there is shown in FIG. 1 a tube 2, whichmay be a glass capillary tube, or other transparent tube, and which maycontain a float/insert 4 made of a plastic, which has a specific gravitythat causes the insert 4 to settle through the red blood cells to thebottom 6, or elsewhere depending on the specific gravity of the float,in the tube 2 when the latter is centrifuged with an anticoagulatedwhole blood sample therein. The fraternal groups of capturematerial-coupled plastic beads or liposomes of different specificgravities or densities may be disposed in a clump 5 in the tube 2. Aplastic cap 10 closes the bottom 6 of the tube 2. The specific gravityof each group of beads will be greater than the specific gravity of thelightest of the red cells, ie, the youngest reticulocytes.

The blood sample is drawn into the tube 2 and, after a suitableincubation period, is centrifuged therein along with the insert 4 andbeads 5. The bead clump 5 disperses in the blood sample during theincubation period, and then settles into distinct bands which form linesin the red cell layer as shown in FIG. 2 during the centrifugation step,while the float/insert 4 settles into and through the red cells R. Thetube 2 will also contain the tagged or labeled analyte-specific bindingmaterial described above.

The white cells W layer out in bands above a red cell/white cellinterface 1. The density-marker bead bands B layer out densimetricallyin the red cell layer. Examination of the bands B will indicate which ofthe bands B have been labeled since the fluorophore labels will bedetectable only in labeled bands.

FIG. 3 shows an alternative form of centrifuge tube which can be used topractice the invention. The tube 12 has a compound funnel-shaped borewith an enlarged open end part 14 and a restricted closed end part 16.It will be appreciated that the tube 12 could also have an end closurecap as previously described, if necessary. The bore is sized so as tocause the majority of the red cells R in the centrifuged blood sample tosettle into the restricted part 16 of the bore, with the majority of theplasma staying for the most part in the enlarged part 14 of the bore.The tagged density-marker bands B disperse in the centrifuged red celllayer. The tube 12 is formed from a transparent glass or plasticmaterial. It will be noted that the embodiment shown in FIG. 3 does notuse a float component.

It will be noted from FIGS. 2 and 3 that the density-marker bands aresufficiently spaced apart that each can be assayed for fluorescence, orother energy emissions, and can even be quantified as set forthhereinafter, without interference from any other bands B. When a bloodsample is assayed, the nature of the red blood cells, ie, the fact thatthey pack when centrifuged in a manner which excludes significantamounts of the plasma, ensures that virtually all of the non-boundlabeled binding material in the tube will end up in the plasma layer,and will not interfere with the procedure. When the sample is beinganalyzed for a particular nucleated cell analyte (ie, a cell having anucleus), the sample may have added thereto a non-specific nuclearcolorant, such as a DNA-specific stain, as for example HOECHST-3542; 7AAD; propidium iodide; SY3-8; or DAPI; or a colorant that stains bothDNA and RNA such as acridine orange or thiozole orange. Any of theaforesaid colorants will stain all nuclei, including the non-analytewhite cells. When the sample is centrifuged, the non-analyte nuceatedcell bands will form above the red cell layer, and the captured analytenucleated cells will, by virtue of the density of their captureparticle, either settle down into the red cell layer or rise above thebuffy coat to form a detectable fluorescence therein. Thus there will betwo areas of fluorescence in the sample, one being the white cell orbuffy coat layer, and the other being the captured nucleated cellanalyte located within the red cell layer or above the white cell layer.The captured analyte cell layer can be quantified in the followingmanner. Assume that the target captured white cell is a particular typeof lymphocyte. The lymphocyte-monocyte cell layer in the buffy coat ismeasured using the QBC® technique, which technique is described in U.S.Pat. No. 4,027,660. The instrument then measures the intensity of thefluorescence emanating from the lymphocyte-monocyte cell layer in thebuffy coat; and then measures the intensity of the fluorescenceemanating from the captured analyte cell layer within the red celllayer. The relative captured analyte cell count is then derived byproportioning the respective fluorescent intensities, and multiplyingthe lymphocyte-monocyte cell count by the resultant proportion decimal.This procedure is enabled by the fact that the red cells will not takeup any of the acridine orange stain. The resultant relative analyte cellcounts will be corrected as follows. The proportion of captured cellfluorescence viz the base line buffy coat cell fluorescence can bedetermined empirically, bearing in mind that the base buffy coat celllayer will be, depending on the size of the assay tubes and capturebodies used in the system, 3 to 5 cells thick, while the captured celllayer will be 1 to 2 cells thick.

Referring now to FIG. 4, a second embodiment of the invention isdisclosed. In this embodiment, the float/insert 4 has one or more bands7 of the analyte-capture binding material coated thereon at axiallyspaced-apart locations on the float/insert 4. Each band is clearlydistinguishable from all of the other bands because of the distancebetween adjacent bands 7. The position of the various bands on thefloat/insert 4 is indicative of the type of analyte-capture materialutilized. In this way the identity of any labeled analytes which bind tothe bands 7 can be confirmed. For example, the lowest band on thefloat/insert can be a binding material specific to Lyme disease; whilethe next lowest band 7 can be a binding material specific to HIV; and soforth. When the binding material-banded float/insert is used inperforming the specimen analysis, the tube, float/insert and sample willpreferably be centrifuged a plurality of times toward opposite ends ofthe tube, so that the float/insert will move back and forth through thesample a number of times. In this manner, the binding material coated onthe float/insert can scavenge the sample for target analytes as thesample passes back and forth through the relatively narrow annulus whichexists between the float/insert and tube, thereby increasing theopportunities for target analytes to be captured on the float/insert.

FIG. 4A shows an embodiment of the invention which uses twofloat/inserts 4 and 4 ' instead of one float/insert. Both of thefloat/inserts may be sized so that once they are placed in the tube,they will not pass each other during the centrifugation steps in thetube. Their cylindrical surfaces will be separated by pointed ends, andwhen a fluorescent float or label is used on floats which centrifugeinto the red cell layer of the blood sample, the pointed ends on thefloats will provide a readily discernible boundary between adjacentfloats because of the ability of the red cells to suppress thefluorescent signal on the pointed ends of the floats. This being thecase, the entire float may be coated with fluorescent material or formedfrom a fluorescent plastic. In the embodiment of FIG. 4A, thefloat/insert 4 has the capture material 7 coated thereon; and thefloat/insert 4' may be devoid of capture material. Both of thefloat/inserts 4 and 4' may or may not be formed from a material whichhas a specific gravity which is greater than the specific gravity of themost dense component in the centrifuge tube, so that both float/insertssink completely through the most dense component in the sample. In caseswhere the floats are desired to end up in the plasma of the centrifugedblood sample, the specific gravity of one or both of the floats will beless than the specific gravity of the lightest of the cells in the bloodsample. When the assembly is centrifuged, the float/insert 4 may settlecompletely into the red blood cell or other density gradient layer, andthe float/insert 4' may settle partially or completely into the red cellor other density gradient layer, and may, when the sample is blood,extend upwardly through the buffy coat to expand the constituent buffycoat cell layers in accordance with the prior art teachings. Both of thefloat/inserts can be used to capture analyte if desired. As notedpreviously, both floats may end up in the plasma layer of the bloodsample if so desired to accommodate an expected low level label signalfrom captured and labeled analyte.

When two or more float/inserts are used, one of the float/inserts can becoated with the label material, as for example, with fluorescein, to adegree which will serve as a reference base which can be used to comparewith the label signal intensity from the captured analyte(s).Alternatively, the reference float may be formed from a fluorescentplastic. In this way, an analyte quantitative count can be obtained. Forexample, if the target analyte were a cell, the coated fluorescencecould be present in a degree that would represent a known count of thetarget cells. Comparison of the level of fluorescence from the capturedanalyte band would then be compared to the level of fluorescence fromthe reference float/insert, and the captured analyte count would then bederived by the instrument software; and the captured analyte count isdisplayed by the instrument.

Referring to FIG. 5, a schematic representation is shown whichillustrates the manner in which the invention can be used to detect thepresence of TSH (thyroid stimulating hormone) molecules in a sample ofanticoagulated whole blood. The density-controlled capture particles 20have a capture agent 22 coupled thereto which is specific to oneepitope, or any specific high affinity binding site, ie the T epitope onthe TSH analyte so as to bind the TSH analyte to the particle 20. Thelabeled binding agent 24 is specific to another epitope, or any specifichigh affinity binding site, ie the H epitope on the analyte, andtherefore will bind to the analyte when added to the blood sample. Theresultant particle-analyte-antibody, or other specific binding substancelabeled complex is shown schematically in FIG. 5.

Referring now to FIG. 6, in cases where one wishes to utilize serum orplasma samples, or even with whole blood samples, when more predictabledensity gradients may be required, such as might be the case if manydensities needed to be separated, then the lower portion of the specimensample tube 2 shown in FIG. 6 could be prefilled with a stable material9, such as gelled Ficoll, which has a greater density than the red bloodcells, and which provides the required density gradient. This densitygradient material, in addition to separating the resident bands, willserve to wash the unbound labeled binding material away from the boundlayers during the centrifugation step. The tube 2 could be provided withan integral internal post 3 which projects upwardly from the bottom wall1 of the tube 2 through the material layer 9. The specimen sample willsettle onto the material layer 9, and the lowermost layer of thespecimen sample, in the case of whole blood, will be the red cell layerR. Since the density marker particles 20 are more dense than the mostdense component of the sample being tested, they will form spaced-apartbands B1, B2, and B3 in the material layer 9. Each band will beinspected for label presence. It will be appreciated that if a closurecap 10 such as is shown in FIGS. 1 and 2 is used, the post 3 could beformed as an integral part of the cap 10. The embodiment shown in FIG. 6can also be used with an anticoagulated whole blood sample wherein nodensity gradient gel is added to the sample, and wherein the post 3projects through the red blood cell and the white blood cell layers inthe centrifuged blood sample.

A general example of the use of the invention to quantify a targetanalyte in a sample is as follows. The physician will identify from theliterature an approximate range of how many molecules or units of atarget analyte can be expected to be found in a known volume sample ofthe biologic fluid being assayed. For example, assume that a patientinfected or exposed to Lyme Disease will be expected to have 50 Lymeanalyte units per milliliter of blood at the most. The physician willadd at least 100 density-marker/capture material coupled units to theblood sample per milliliter being sampled, and will also add at least100 labeled binding material units per milliliter of sample to thecontainer. Since there are an excess of capture sites and labeledparticles in the sample as compared to the maximum number of analyteunits expected to be found in the sample, the degree or intensity oflabel emission from the Lyme bead band will be proportional to thenumber of Lyme analytes which are actually present in the sample. Aquantification of the Lyme analyte in the blood can thus be approximatedby measuring the emission intensity. The key to the quantificationprocedure is to provide a functional excess of capture sites and labeledbinding material units in the sample as compared to the maximum numberof analyte units which can be expected to be found in the sample. Onemay still be able to quantitate the analyte even if the bound labeledbinding material units are present in molar amounts less than theanalyte, provided that there exists a mathematical relationship betweenthe amount of analyte present and the amount of analyte eventuallycaptured by the density marker/labeled binding material couples.

An example of a type of another target analyte that can be detected bythe invention is a subset of lymphocyte blood cells. Normal human bloodcontains a relatively predictable amount of a subset of lymphocyte cellswhich have a surface epitope, or any specific high affinity binding siteidentified as CD4. It has been verified that the CD4lymphocytepopulation in the blood of one who has been infected by HIV eventuallydeclines, before the infected individual demonstrates symptoms of theinfection. Antibodies have been identified that are specific to theCD4epitope, or any specific high affinity binding site of theselymphocytes, and these antibodies can be attached to both the capturedensity marker and to the label. When cells are the analyte, rather thanmolecules, the epitope, or any specific high affinity binding site onthe cellular analyte to which the density marker bonds can be the sameor a different epitope, or any specific high affinity binding site thanthat the labeled binding material bonds. The reason that the cellepitope, or any specific high affinity binding site can be the same forthe density marker capture substance and the labeled binding substanceis because of the very large size of a cell as compared to an analytewhich is generally a molecule. The principal governing this factor isillustrated in FIG. 7. Referring to FIG. 7, the cellular analyte isdenoted by the numeral 26. The cell 26 will have a number of surfaceepitope, or any specific high affinity binding sites 28 and 30, one ofwhich is CD4. The epitope, or any specific high affinity binding sites28 and 30 will be specific to different binding material moieties. Thenumeral 20 indicates the density marker, and the numeral 22 indicatesthe binding material moiety bonded to the density marker 20. In the caseillustrated in FIG. 7, the binding material is specific to the cellepitope, or any specific high affinity binding site 28 and will bondthereto. The surface area of the cell 26 sufficiently large, as comparedto the binding material moieties 22 and 24 so as to present a very largenumber of available epitope, or any specific high affinity binding sites28 and 30, so as to allow the labeled binding material moiety 24 to bespecific to an epitope, or any specific high affinity binding site 30which is different from the density marker epitope, or any specific highaffinity binding site 28, or to be specific to the same epitope, or anyspecific high affinity binding site 28. Therefore, when a cell is theanalyte, the density marker binding material and the labeled bindingmaterial may be specific to the same, or to a different, epitope, or anyspecific high affinity binding site on the cellular analyte. Thus theassay of CD4 lymphocytes may be accomplished with densitymarker/CD4antibody couples, and labeled CD4antibodies. Alternatively,other epitope, or any specific high affinity binding sites on theCD4cells may be used for the density markers or for the labeled bindingmaterial moieties. Once the CD4(or any other cellular analyte)lymphocyte/density-marker/labelled binding material complexes areformed, they can be detected and quantified in the manner describedabove.

Alternatively, the labelled binding material could be omitted, and ageneral DNA stain, such as propidium iodide could be used toquantitatively stain the cell nuclei. In such a case, the totalfluorescence signal in the capture area would be directly proportionalto the number of captured cells.

In the CD4cell assay, an area of the float having not captured cells orfluorescence may be needed to provide a background count in someinstances. This will take into account light scatter and fluorescencefrom the layer of plasma (which will contain the label such as acridineorange) that surrounds the float and lies between the float and the tubewall. Yet another target analyte that can be detected by the inventionis hematopoietic progenitor blood cells. Hematopoietic progenitor bloodcells are contained within a cell population recognized with themonoclonal antibody, or other specific binding substance CD34. Thesecells constitute approximately 1% of human bone marrow cells, and can befound in bone marrow, peripheral blood, and cord blood. In peripheralblood of normal individuals the number of CD34cells is approximately 0.1to 10 per microliter (0.01% of leukocytes). This CD34 cell populationnumber increases upon treatment with chemotherapy and/or growth factors,and numbers as high as 1,000 per microliter have been observed. In asampling tube holding 100 μl of blood, 10 to 500 CD34 cells are thuspresent in normal individuals, and this number can increase to100,000/μl in patients being treated for cancer. High dose chemotherapyhas shown to be beneficial for cancer patients. Ablative chemotherapyhowever results in pancytopenia (a general decrease in all cell lines)which has to be rescued by reinfusion of hematopoietic stem cells. Sincehematopoietic stem cells are contained within the CD34 cell population,it is essential to quantify the number of CD34 cells before they arereinfused into the patients.

Antibodies have been identified that are specific to the CD34 epitope,or any specific high affinity binding site of these blood cells, andthese CD34 antibodies can be used to assay the CD34 cell population inthe same general manner as described above for the CD4cells. Once theCD34 cell/density-marker/labelled antibody, or other specific bindingsubstance complexes are formed, they will be detected and quantified inthe manner described above.

It will be appreciated that the invention has been described inconnection with blood diagnosis, but the invention is also applicable todiagnose other biological fluids for the presence or absence of highlyspecific complementary couples found in such other biological fluids. Aswith the analysis of plasma, when a biological fluid other than wholeblood is assayed, the centrifugation step can be performed in thedensity gradient fluid, such as Ficoll gel, as noted above, which willnot mix with the aqueous phase of the biological fluid, and will promotedensimetric separation of the bands in the density gradient fluid, withconcurrent washing by the gradient fluid of the densitymarkers, toensure separation of all non-bound label from the bands. This eliminatesnon-bound label interference with quantification of the labeled bands.The inherent washing of non-bound label from labeled cells when wholeblood is being tested, and when a non-cellular fluid is being tested ingelled Ficoll, which washing occurs during the centrifugation step,eliminates the separate washing steps required by the prior art, andprevents unbound label from interfering with the accuracy of theprocedure. This inherent washing is an important advantage of thisinvention. The labeled captured analytes can be quantified by measuringthe intensity of label emission eminating from the captured analyte bandor bands in the sample. An instrument similar to that shown in U.S. Pat.No. 4,558,947 which is modified to include a light-intensity-quantifiercan be used to perform analyte presence or absence, and/or analytequantifications, as well as to perform buffy coat analysis.

Since many changes and variations of the disclosed embodiments of theinvention may be made without departing from the inventive concept, itis not intended to limit the invention otherwise than as required by theappended claims.

What is claimed is:
 1. A method for detecting a target analyte in abiologic fluid sample in a transparent tube, said method comprising thesteps of:a) adding one or more capture bodies to the sample, whichcapture bodies have a predetermined specific gravity, each capture bodybeing coupled with a binding material to form one or more capture bodycouples which are specific to a binding site on the target analyte; b)adding to said sample, labeled binding material moieties which arespecific to a binding site on said target analyte to form a capture bodybinding material couple/labeled binding material moiety sample mixture;c) incubating the capture body binding material couple/labeled bindingmaterial moiety sample mixture; d) densimetrically displacing thecapture bodies into a predictable region in the sample mixture; and e)determining if any capture bodies in the sample exhibit the presence ofthe labeled binding material, and therefore the presence of the targetanalyte in the sample.
 2. The method of claim 1 further comprising thestep of displacing unbound labeled antibodies from the capture bodiesduring the step of densimetric separation.
 3. The method of claim 2wherein the fluid sample is anticoagulated whole blood, and wherein thecapture bodies have a specific gravity that is less than the specificgravity of the heaviest of any red cells in the blood sample.
 4. Themethod of claim 3 wherein the capture bodies have a specific gravitywhich is greater than the specific gravity of the heaviest of the anyred cells.
 5. The method of claim 3 wherein said step of displacing isperformed by the cells in the whole blood sample.
 6. The method of claim1 wherein the fluid sample is an aqueous based biological sample.
 7. Themethod of claim 6 further comprising the step of displacing unboundlabeled antibodies from the capture bodies during the step ofdensimetric separation.
 8. The method of claim 7 wherein said step ofdisplacing is performed by providing a label-immiscible density gradientmaterial in the tube, into which density gradient material thedensity-markers settle.
 9. The method of claim 1 wherein thedensity-markers are densimetrically separated into a part of the tubehaving an internal sample-occupying portion which is less in crosssectional area than the cross sectional area of the remainder of thetube.
 10. The method of claim 9 wherein said portion of the tube isformed by positioning an axially elongated insert in the tube.
 11. Themethod of claim 9 wherein said portion of the tube is formed by alocalized constriction in the tube.
 12. A method for detecting one ormore different target analytes in a biologic fluid sample in atransparent tube, said method comprising the steps of:a) adding capturebodies to the sample, there being one group of capture bodies for eachtarget analyte suspected to be in the sample, each group of capturebodies having a different specific gravity from each other group ofcapture bodies, and each capture body in each group thereof beingcoupled with a binding material which is specific to a first epitope orother binding site on one of the target analytes, whereby each of thedifferent capture body/binding material couple groups is specific to adifferent one of the suspected target analytes; b) adding to said samplelabeled antibodies or other binding material which is specific toanother epitope or other binding site on each of said analytes to form acapture body/binding material couple and labeled binding material samplemixture; c) incubating the capture body/binding material and labeledbinding material sample mixture; d) densimetrically separating thecapture bodies into one or more distinct bands in the sample mixture;and e) determining which, if any of said bands exhibit the presence of alabeled antibody or other binding material, and therefore the presenceof a target analyte.
 13. The method of claim 12 further comprising thestep of displacing unbound labeled antibodies or other labeled bindingmaterial from the capture bodies during the step of densimetricseparation.
 14. The method of claim 13 wherein the fluid sample isanticoagulated whole blood, and wherein the capture bodies have aspecific gravity that is greater than the specific gravity of theheaviest of red cells in the blood sample.
 15. The method of claim 14wherein the capture bodies have a specific gravity which is less thanthe specific gravity of the heaviest of the red cells.
 16. The method ofclaim 14 wherein said step of displacing is performed by cells in thewhole blood sample.
 17. The method of claim 12 wherein the fluid sampleis an aqueous based biological sample.
 18. The method of claim 17further comprising the step of displacing unbound labeled antibodies orother binding material from the capture bodies during the step ofdensimetric separation.
 19. The method of claim 18 wherein said step ofdisplacing is performed by providing a label-immiscible density gradientmaterial in the tube, into which density gradient material the capturebodies settle.
 20. The method of claim 12 wherein the capture bodies aredensimetrically separated into a part of the tube having an internalsample-occupying portion which is less in cross sectional area than thecross sectional area of the remainder of the tube.
 21. The method ofclaim 20 wherein said portion of the tube is formed by positioning anaxially elongated insert in the tube.
 22. The method of claim 20 whereinsaid portion of the tube is formed by a localized constriction in thetube.
 23. A method for detecting one or more different target analytesin an anticoagulated whole blood sample in a transparent tube, saidmethod comprising the steps of:a) adding capture bodies to the sample,there being one group of capture bodies for each target analytesuspected to be in the sample, each group of capture bodies having adifferent specific gravity from each other group of capture bodies, withall of the capture bodies having a specific gravity that will ensurethat said capture bodies will settle into the red cell layer of theblood upon centrifugation of the sample in the tube, and each capturebody in each group thereof being coupled with a binding material whichis specific to one of the target analytes, whereby each of the differentcapture body/binding material couple groups is specific to a differentone of the suspected target analytes; b) adding to said sample labeledantibodies or other binding material specific to said target analytes soas to form a capture body/binding material and labeled binding materialsample mixture; c) incubating the capture body/binding material andlabeled binding material sample mixture; d) centrifuging the sample soas to aggregate the capture bodies into one or more distinct bands; ande) determining which, if any of the bands exhibit the presence of alabeled antibody or other binding material, and therefore the presenceof a target analyte.